Duchenne muscular dystrophy (DMD) results from mutations in the dystrophin gene and is among the most common genetic disorders. Dystrophin nucleates the assembly of a membrane protein complex known as the dystrophin-glycoprotein complex (DGC). Dystrophin links the subsarcolemmal cytoskeleton to the extracellular matrix via binding to v-actin at its N-terminal and central rod domain, and via binding to the DGC at more distal C-terminal domains. The DGC members (3-dystroglycan and the sarcoglycans span the sarcolemma and bind a-dystroglycan, which anchors the complex to laminin in the extracellular matrix. Despite a wealth of information on the function of dystrophin and the core components of the DGC, little is known about the function of DGC members that bind the dystrophin C-terminal domain, namely the dystrobrevins (Db), syntrophins and neuronal nitric oxide synthase (nNOS). These proteins are thought to play signaling roles in muscle, in contrast to the predominantly mechanical role of dystrophin. Assembly of the DGC by the non-muscle dystrophin isoform Dpi 16, which does not bind actin, has no effect in dystrophin-deficient mdx mice (a model for DMD). However, Dp116 prevents early lethality and leads to normal muscle and body mass in dystrophin:utrophin double knockout (mdx:utrn-/-) mice. We propose to study the structural and functional determinants of the DGC that rescue early lethality in mdx:utrn-/- mice. We will also explore the function of aDb, with a focus on the poorly studied aDb-3 isoform that lacks a dystrophin-binding site. These studies will identify how aDb-3 associates with the DGC, and whether it can ameliorate any functional deficiencies in skeletal or cardiac muscles of dystrophic aDb-/- mice. Finally, we will generate novel mini- and micro-dystrophins constructs that might lead to more complete assembly of the DGC in muscle. Current micro-dystrophins are of great interest for developing gene therapy methods yet they do not fully restore strength to dystrophic muscles. Improved mini-/micro-dystrophins may allow a better understanding of how the DGC is assembled and may prove more suitable for gene replacement strategies. This proposal explores the function of dystrophin and the dystrophin-glycoprotein complex, proteins that when defective lead to various types of muscular dystrophy. Gaining a better understanding of how these proteins maintain normal muscle function is important for developing therapies for the muscular dystrophies